Papillomaviruses induce proliferative epithelial lesions and can only undergo vegetative replication in terminally differentiated keratinocytes. This has hampered the study of the complete viral life cycle because of the difficulties in generating a differentiated stratified epithelium in tissue culture. Using a combination of organotypic raft cultures and xenografts on nude mice we have developed a system in which we can generate fully differentiated bovine epithelium in which BPV-1 can replicate and produce infectious viral particles. Organotypic cultures are established with bovine keratinocytes plated on a collagen raft containing BPV-1 transformed fibroblasts. The keratinocyte monolayer is infected with virus particles isolated from a bovine wart or transfected with cloned viral DNA. Several days after lifting the rafts to the air interface they are grafted on nude mice. After approximately six to eight weeks, large xenografts are produced that exhibit a hyperkeratotic epithelium with a large dermal fibroma. Virus particles can be isolated from these lesions and quantitated by a focus forming assay on mouse cells in culture. We have optimized this system so that xenografts can be very efficiently obtained.Studies are in progress to analyze the ability of papillomavirus genomes with mutations in specific genes to undergo the complete viral life-cycle. Mutations currently being tested are in the E2 and E4 genes. E2 is a multifunctional protein important for transcription and DNA replication. No function has been assigned to the E4 protein. - Papillomavirus, DNA replication, transcription, virology, cancer, gene transfer, keratinocyte differentiation